rabbit polyclonal anti trpa1  (Novus Biologicals)

Journal: Pain Research & Management

Article Title: Exploring the Analgesic Initiation Mechanism of Tuina in the Dorsal Root Ganglion of Minor CCI Rats via the TRPV1/TRPA1-cGMP Pathway

doi: 10.1155/2024/2437396

Figure Lengend Snippet: Primary and secondary antibodies used for Western blotting.

Article Snippet: Primary , Rabbit anti-TRPA1 polyclonal IgG , 1 : 1000 , Proteintech Group, Chicago, USA.

Techniques: Western Blot, Concentration Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels

doi: 10.1152/ajpheart.00380.2023

Figure Lengend Snippet: Sensitivity (EC 50 ) of 0.1–300 µM α-pinene-, myrtenol-, and verbenol-induced relaxations in PE-precontracted murine SMA in the presence and absence of treatments or SMA of transgenic mice

Article Snippet: Immunofluorescence microscopy was performed with rabbit polyclonal antibody against TRPA1 (1:200 dilution; Alomone Labs Cat. No. ACC-037, RRID:AB_2040232) and Alexa Fluor 647 goat anti-rabbit secondary antibody (1:400 dilution; Invitrogen; 21244) with or without a blocking peptide of the TRPA1 antibody.

Techniques: Transgenic Assay, Control

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels

doi: 10.1152/ajpheart.00380.2023

Figure Lengend Snippet: Efficacy (E max ) of 300 µM α-pinene-, myrtenol-, and verbenol-induced relaxations in PE-precontracted SMA (male and female) with and without inhibitor treatments or SMA of transgenic mice

Article Snippet: Immunofluorescence microscopy was performed with rabbit polyclonal antibody against TRPA1 (1:200 dilution; Alomone Labs Cat. No. ACC-037, RRID:AB_2040232) and Alexa Fluor 647 goat anti-rabbit secondary antibody (1:400 dilution; Invitrogen; 21244) with or without a blocking peptide of the TRPA1 antibody.

Techniques: Transgenic Assay, Control

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels

doi: 10.1152/ajpheart.00380.2023

Figure Lengend Snippet: Mechanisms of myrtenol- and verbenol-induced relaxation in C57BL/6J superior mesenteric artery (SMA). Representative traces ( A ) and normalized data ( B ) of myrtenol- and verbenol-stimulated (0.1–300 µM) relaxation of 10 µM phenylephrine (PE)-precontracted SMA ( n = 3,3 C57BL/6J mice) compared with α-pinene-induced relaxation ( B only). Representative traces ( C ) and summarized data ( D ) of 1–300 µM myrtenol-stimulated relaxation of 10 µM PE-precontracted wild-type (WT) SMA without [control; dimethyl sulfoxide (DMSO)] and with transient receptor potential ankyrin-1 (TRPA1) inhibitor, A-967079 (1 µM) ( n = 4, 4 mice, respectively). Representative traces ( E ) and summarized data ( F ) of myrtenol-stimulated relaxations of PE-precontracted SMA of WT and TRPA-null mice ( n = 7, 3 mice, respectively). Values are means ± SE; n , 3–7 mice per group. For comparison of two concentration-dependent response curves, a two-way ANOVA with repeated measures and Bonferroni all pairwise post hoc test was used.

Article Snippet: Immunofluorescence microscopy was performed with rabbit polyclonal antibody against TRPA1 (1:200 dilution; Alomone Labs Cat. No. ACC-037, RRID:AB_2040232) and Alexa Fluor 647 goat anti-rabbit secondary antibody (1:400 dilution; Invitrogen; 21244) with or without a blocking peptide of the TRPA1 antibody.

Techniques: Control, Comparison, Concentration Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels

doi: 10.1152/ajpheart.00380.2023

Figure Lengend Snippet: Immunofluorescent localization of transient receptor potential ankyrin-1 (TRPA1) in C57BL/6J superior mesenteric artery (SMA). Formalin-fixed, paraffin-embedded sections of murine male wild-type (WT) SMA were stained with hematoxylin and eosin (H&E; A ); TRPA1 antibody (Ab) only (green, arrows point to endothelium; B ); TRPA1 Ab (green), isolectin Ab (red, endothelium), and 4′,6-diamidino-2-phenylindole (DAPI) (blue, nucleus; C ) or TRPA1 Ab, TRPA1 blocking peptide, isolectin Ab, and DAPI ( D ). L, lumen. All images were taken at ×200 magnification (scale bar = 100 μm). Summarized data α-pinene ( E )- and calcitonin gene-related peptide (CGRP; F )-stimulated relaxations of PE-precontracted SMA of WT mice without [control; dimethyl sulfoxide (DMSO)] and with CGRP receptor antagonist, SB-268262 (1 µM) ( n = 3,5 mice, respectively). Values are means ± SE; n , 3–5 mice per group. For comparison of two concentration-dependent response curves, a two-way ANOVA with repeated measures and Bonferroni all pairwise post hoc test was used. * P < 0.05; treatment vs. control.

Article Snippet: Immunofluorescence microscopy was performed with rabbit polyclonal antibody against TRPA1 (1:200 dilution; Alomone Labs Cat. No. ACC-037, RRID:AB_2040232) and Alexa Fluor 647 goat anti-rabbit secondary antibody (1:400 dilution; Invitrogen; 21244) with or without a blocking peptide of the TRPA1 antibody.

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Blocking Assay, Control, Comparison, Concentration Assay

Journal: BMC Immunology

Article Title: TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

doi: 10.1186/s12865-023-00549-0

Figure Lengend Snippet: TRPA1 regulates the activation of macrophages in Hsp90-inhibited condition. RAW 264.7 cells were treated with different conditions of LPS/PMA, 17-AAG, HC-030031, or AITC and harvested at 12 h. FC histogram depicting fold change in MFI of MHCII ( A , D ), CD80 ( B , E ), and CD86 ( C , F ) of macrophages treated with LPS (500ng/ml, A-C) or PMA (100 ng/ml, D-F) along with their respective bar graphs. The data represent the mean ± SD of at least three independent experiments. One-way ANOVA has been performed to find statistical significance among groups. Differences between groups with a p-value less than 0.05 were considered statistically significant (ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: Rabbit polyclonal antibody against extracellular TRPA1 with specific blocking peptide [TRPA1, INSTGIINETSDHSE] was obtained from Alomone Laboratories (Jerusalem, Israel).

Techniques: Activation Assay

Journal: BMC Immunology

Article Title: TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

doi: 10.1186/s12865-023-00549-0

Figure Lengend Snippet: TRPA1 regulates the nitric oxide (NO) production in Hsp90-inhibited monocytes/macrophages. RAW 264.7 and THP-1 cells were treated with different conditions of LPS/PMA, 17-AAG, HC-030031, or AITC, and the supernatant was collected at 6 h,12 h, and 24 h. Bar graph depicting nitric oxide production from RAW 264.7 cells treated with LPS (500 ng/ml) ( A ) or PMA (100 ng/ml) ( C ) or THP-1 macrophages treated with LPS (500 ng/ml) ( B ). The data represent the mean ± SD of at least three independent experiments. One-way/two-way ANOVA was performed to find statistical significance among groups. Differences between groups with a p-value less than 0.05 were considered statistically significant (ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: Rabbit polyclonal antibody against extracellular TRPA1 with specific blocking peptide [TRPA1, INSTGIINETSDHSE] was obtained from Alomone Laboratories (Jerusalem, Israel).

Techniques:

Journal: BMC Immunology

Article Title: TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

doi: 10.1186/s12865-023-00549-0

Figure Lengend Snippet: TRPA1 regulates the pro-inflammatory cytokine production in Hsp90-inhibited monocytes/macrophages. RAW 264.7 or THP-1 cells were subjected to different conditions of LPS/PMA, 17-AAG, HC-030031, or AITC, and the supernatant was collected at 6 and 24 h and assessed for cytokine profile. Bar graph representing IL-6 ( A , C , E ) and TNF ( B , D , F ) levels in RAW 264.7 cells stimulated with either LPS (500 ng/ml) ( A ) / PMA (100 ng/ml) ( C ) and THP-1 macrophages stimulated with LPS (500ng/ml) ( B ). The data represent the mean ± SD of three independent experiments. One-way/two-way ANOVA was performed to find statistical significance among groups. Differences between groups with a p-value less than 0.05 were considered statistically significant (ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: Rabbit polyclonal antibody against extracellular TRPA1 with specific blocking peptide [TRPA1, INSTGIINETSDHSE] was obtained from Alomone Laboratories (Jerusalem, Israel).

Techniques:

Journal: BMC Immunology

Article Title: TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

doi: 10.1186/s12865-023-00549-0

Figure Lengend Snippet: TRPA1 regulates the Hsp90 inhibition-mediated downregulation of MAPK signaling protein phosphorylation in LPS-stimulated monocytes/macrophages. RAW 264.7 cells were subjected to different conditions of LPS (500 ng/ml), 17-AAG, HC-030031, and AITC harvested at 15 min and assessed for intracellular signaling proteins p38-MAPK, ERK 1/2, SAPK-JNK and their respective phosphorylated proteins via western blot. ( A ) Western blot images from the samples represent p38-MAPK, ERK 1/2, SAPK-JNK, and their respective phosphorylated proteins. Bar graph representing the fold change in band intensity of phospho-proteins p-p38-MAPK ( B ), p-ERK 1/2 ( C ), and p-SAPK-JNK ( D ) normalized to the corresponding GAPDH controls. The data represent the mean ± SD of three independent experiments. Differences between groups with a p-value less than 0.05 were considered statistically significant (ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: Rabbit polyclonal antibody against extracellular TRPA1 with specific blocking peptide [TRPA1, INSTGIINETSDHSE] was obtained from Alomone Laboratories (Jerusalem, Israel).

Techniques: Inhibition, Phospho-proteomics, Western Blot

Journal: BMC Immunology

Article Title: TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

doi: 10.1186/s12865-023-00549-0

Figure Lengend Snippet: TRPA1 regulates apoptosis in Hsp90-inhibited and LPS/PMA-stimulated macrophages. RAW 264.7 cells were treated with different conditions of LPS (500 ng/ml)/PMA (100 ng/ml), 17-AAG, HC-030031, and AITC. Cells were harvested at 5 and 24 h. Heat-killed cells were used as a positive control. ( A ) FC dot plots representing the percentage of positive cells for Annexin V and 7-AAD at 24 h. Double-positive cells were considered either dead or late apoptotic. Representative bar graphs of sample stimulated with LPS (500 ng/ml) ( B )/PMA (100 ng/ml) ( C ). ( D , E ) Western blot image and bar graph representing fold change in band intensity for cleaved caspase 3 ( D , E ) for the respective samples. The data represent the mean ± SD of three independent experiments. Differences between groups with a p-value less than 0.05 were considered statistically significant (ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: Rabbit polyclonal antibody against extracellular TRPA1 with specific blocking peptide [TRPA1, INSTGIINETSDHSE] was obtained from Alomone Laboratories (Jerusalem, Israel).

Techniques: Positive Control, Western Blot

Journal: BMC Immunology

Article Title: TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

doi: 10.1186/s12865-023-00549-0

Figure Lengend Snippet: TRPA1 regulates intracellular calcium influx in LPS-stimulated and Hsp90-inhibited macrophages. RAW 264.7 cells were treated with Fluo-4 AM and assessed via FC for intracellular calcium levels upon combinatorial treatment with either ionomycin, vehicle (DMSO), vehicle + LPS, LPS + 17-AAG, LPS + 17-AAG + HC-030031, and LPS + 17-AAG + AITC. ( A ) Time-lapse kinetics of intracellular calcium influx. The X-axis of the flow cytometric plots represent approximately 200 s and ‘↓’ represents the addition of reagents/modulators to stimulate cells. ( B ) Representative line graph depicting fold changes in mean Fluo-4 intensity. The data represent the mean ± SD of three independent experiments.

Article Snippet: Rabbit polyclonal antibody against extracellular TRPA1 with specific blocking peptide [TRPA1, INSTGIINETSDHSE] was obtained from Alomone Laboratories (Jerusalem, Israel).

Techniques:

Journal: BMC Immunology

Article Title: TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro

doi: 10.1186/s12865-023-00549-0

Figure Lengend Snippet: A proposed comprehensive working model. A detailed working model depicting the role of TRPA1 in 17-AAG mediated regulation of inflammation in LPS or PMA stimulated macrophages. TRPA1 is modulated upon LPS/PMA stimulation ( A ). pro-inflammatory responses including IL-6, TNF, MHCII, CD80/86, NO, intracellular calcium, and intracellular signaling proteins p38-MAPK, p-ERK 1/2, p-SAPK-JNK are significantly upregulated with LPS ( A.1 ) or PMA stimulation ( A.2 ). Upon administration of 17-AAG along with LPS or PMA, the pro-inflammatory responses are downregulated ( C ). Inhibition of TRPA1 via HC-030031 with 17-AAG and LPS or PMA administration reverses the pro-inflammatory responses, intracellular signaling proteins p38-MAPK, p-ERK 1/2, p-SAPK-JNK back to the LPS or PMA stimulated levels with a further diminished intracellular calcium level ( B ). Activation of TRPA1 via AITC and 17-AAG in LPS or PMA stimulated macrophages by impairing the proinflammatory responses, intracellular calcium, and intracellular signaling proteins p-p38-MAPK, p-ERK 1/2, p-SAPK-JNK to a greater extent exhibiting an anti-inflammatory property ( D ). The pro-inflammatory responses are represented according to the color code.

Article Snippet: Rabbit polyclonal antibody against extracellular TRPA1 with specific blocking peptide [TRPA1, INSTGIINETSDHSE] was obtained from Alomone Laboratories (Jerusalem, Israel).

Techniques: Inhibition, Activation Assay

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: Structure of a subunit of the homotetrameric TRPA1 receptor. TM = transmembrane domain; intra = intracellular space; extra = extracellular space; N = N-terminus of the peptide, C = C-terminus of the peptide. Symbols of the highlighted amino acids: C = cysteine; G = glutamine; I = isoleucine; K = lysine; S = serine; T = threonine; Y = tyrosine.

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques:

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: Process of the TRPA1 PCR-based site-directed mutagenesis, with the example of C621A mutation. (A) Starting from the wild-type TRPA1 coding pT31-hTRPA1 plasmid, in the first PCR, the two halves of the cloning region were separately amplified by a mixed pair of mutation primer and cloning primer (in this case: Cys-F2 with C621A-R, and C621A-F with Cys-R2). (B) In the second PCR, the two halves were joined and amplified by Cys-F2 and Cys-R2 cloning primers. (C) Mutation-carrying complete PCR product and the original pT31-hTRPA1 plasmid were sequentially digested by BstEII and BbvCI unique restriction endonucleases. (D) Mutation-carrying PCR product was ligated into the “empty” plasmid, creating the mutant TRPA1 coding expression vector. CMV–cytomegalovirus promoter; AmpR–ampicillin resistance cassette; Kan/NeoR–kanamycin and neomycin resistance cassette; ColE\Ori– E. coli specific origin of replication; blue region–TRPA1 cDNA; orange–primer sites; black-lettered bases–codon of the 621st cysteine; red-lettered bases–codon of alanine (C621A mutation); purple-lettered bases–DNA sticky end after BstEII cleaving; pink-lettered bases–DNA sticky end after BbvCI cleaving.

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Mutagenesis, Plasmid Preparation, Cloning, Amplification, Expressing

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: Structure of tested TRPA1 agonists. Organic polysulfides, dimethyl trisulfide (DMTS), diallyl trisulfide (DATS), and diallyl disulfide (DADS), are reversible electrophile agonists. JT010 is a very potent and selective irreversible electrophile TRPA1 agonist. Allyl isothiocyanate (AITC) or mustard oil is a potent natural reversible electrophile TRPA1 agonist. Carvacrol is a non-electrophile TRPA1 agonist with different binding sites on TRPA1, and we used it as a control activator. Illustration is made by ChemDraw Direct (RRID: SCR_016768).

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Binding Assay, Control

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: Covalently docked binding position of DMTS to the native holo TRPA1 (attached covalently to C621) and the non-covalently docked binding position of DMTS to the alanine mutant TRPA1 (located further to the left). DMTS is shown as all atom representation sticks (teal). The protein is shown as gray cartoon. A-loop (see Results) is labeled.

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Binding Assay, Mutagenesis, Labeling

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: Covalent docking of DMTS, DADS, and DATS with FITTED to the three cysteines forming the nucleophilic binding cavity in the intracellular TRPA1 (6pqp holo structure) agonist binding site. Non-covalent docking of the same ligand to the C621A mutant receptor. All calculated binding free energy values are in kcal/mol.

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Binding Assay, Mutagenesis

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: DMTS dose–effect curves in TRPA1 mutant variants, compared to the effect of 1 mM carvacrol. Fluo-4 calcium-sensitive fluorescence was measured by flow cytometry in different TRPA1 variants, using increasing DMTS concentration, compared to the fluorescent signal of 1 mM carvacrol (100%). The single mutation of C621, C641, and C834 decreased the effect of DMTS in TRPA1, but C621A/C641A double mutant and C621A/C641A/C665A triple mutant showed insensitivity to DMTS, indicating that these amino acids are involved in the binding site of the organic polysulfides. The combined double mutation of C727 and C834 (C727A/C834A) made no significant difference from the wild-type TRPA1, suggesting that they are not parts of the binding site of the organic polysulfides. Mean ± SEM, N = 2-4 (number of measurements per TRPA1 variation), n > 10000 (number of cells per measurement).

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Mutagenesis, Fluorescence, Flow Cytometry, Concentration Assay, Binding Assay

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: TRPA1 activity measured by calcium-sensitive fluorescence flow cytometry. The different TRPA1 variant-expressing CHO cells were stained by Fluo-4 calcium-sensitive fluorophore. TRPA1 activation causes calcium influx, and therefore increases the intensity of fluorescence, which was measured by flow cytometry. The signal intensity was compared to the effect of 100 µM carvacrol, which was used as a non-electrophilic control agonist. Relatively high concentration (100 µM) of organic polysulfides (DMTS, 90% DADS and 63.5% DATS) showed only slight changes in their receptor-activating effect in the case of TRPA1 single mutants (C621A, C641A, and C665A), but their effect was completely eliminated in the triple-mutant TRPA1 variant (C621A/C641A/C665A). The effect of JT010 was eliminated even in the C621A single mutant, and the other two single mutants also showed a tendency for decreased JT010 sensitivity. Diagram shows the means ± SEM, two-way ANOVA with Bonferroni’s multiple comparison test, significance compared to WT TRPA1: * p < 0.0001, WT TRPA1 replication experiments (N = 4-12), mutant TRPA1 replication experiments (N = 2-5), sample size ( n = 4–19), cell number per sample ( n > 2000).

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Activity Assay, Fluorescence, Flow Cytometry, Variant Assay, Expressing, Staining, Activation Assay, Control, Concentration Assay, Mutagenesis, Comparison

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: TRPA1 activity measured by radioactive Ca-45 liquid scintillation counting. The radioactive Ca-45 uptake due to TRPA1 activation was measured by liquid scintillation counting and compared to the effect of 100 µM carvacrol. Out of the three TRPA1 single mutants, C612A shows the highest decrease in sensitivity to electrophilic agonists, indicating its key role in agonist binding. Triple mutation eliminated the effect of electrophilic agonists. Diagram shows the individual values and means ± SEM, two-way ANOVA with Bonferroni’s multiple comparison test, significance compared to WT TRPA1: * p < 0.005, ** p < 0.0001, n = 3–6

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Activity Assay, Activation Assay, Binding Assay, Mutagenesis, Comparison

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet: Effect of the polysulfides on the WT and on the cysteine mutant TRPA1 using the whole -cell patch-clamp technique. (A) Representative whole-cell patch-clamp measurement of a WT TRPA1-expressing CHO cell. Until the continuous line, Ca2+ free control solution was applied, and between the continuous and the first dotted line, the effect of 100 µM DMTS is shown. Between the two dotted lines, and after the second dotted line, we can see the effects of the two positive controls (carvacrol and HC-030031, respectively). The red symbols represent the peak currents at +50 mV, while the blue ones are the peak currents at -50 mV command voltage. (B) Representative whole-cell measurements regarding the WT, C621A, C641A, C665A, C621A/C641A, and C621A/C641A/C665A populations (saturated traces). The green, blue, purple, and black solid lines were measured in the presence of 100 µM carvacrol, 100 µM DMTS, 50 µM HC-030031, and Ca2+-free control solution, respectively. The horizontal dashed line represents 0 pA current. The voltage protocol is shown with a solid red line as an inset on the top left panel. The fluctuation of the effect of carvacrol correlated to and indicated the overall TRPA1 response rate. (C–E) Bar charts of the activated current ratios by the application of 100 µM DMTS, 90% DADS, and 63.5% DATS, respectively. The colored bars represent the WT and the C621A, C641A, C665A, C621A/C641A, and C621A/C641A/C665A mutant TRPA1 (N transfection ≥2 and n cell ≥5 for each population). Black dots indicate individual measurements, and bar chart height and error bars show mean ± SEM. Asterisks represent significant difference (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001).

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Mutagenesis, Patch Clamp, Expressing, Control, Transfection

Journal: Frontiers in Physiology

Article Title: Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

doi: 10.3389/fphys.2023.1180896

Figure Lengend Snippet:

Article Snippet: The expression of TRPA1 variants was tested by immunohistochemistry, using polyclonal anti-human TRPA1 rabbit IgG primary antibody (Thermo Fisher OST00061W, RRID: AB_2207890) and peroxidase-linked polyclonal anti-rabbit IgG goat IgG secondary antibody (Thermo Fisher Scientific 31460, RRID: AB_228341), and stained by the 3,3′-diaminobenzidine (DAB) Liquid Substrate System (Sigma-Aldrich D7304).

Techniques: Sequencing, Mutagenesis, Recombinant, Expressing, Modification, Solvent, Gas Chromatography, Mass Spectrometry, Polymerase Chain Reaction